Lipids such as cholesterol, phospholipids, neutral fats (triglycerides) and the like are contained in blood, and determination of cholesterol among them is widely used as a screening test for arteriosclerosis, hepatic diseases, diabetes mellitus or transient lipid metabolism disorders. Cholesterol forms complexes with proteins, phospholipids, triglycerides and the like in blood and exists as lipoproteins. The lipoproteins include high density lipoprotein (HDL), low density lipoprotein (LDL), very low-density lipoprotein (VLDL) and modified lipoproteins and the like, and analysis of each lipoprotein is useful for clinical diagnoses.
Several methods have been proposed as the method for separating lipoproteins by high performance liquid chromatography techniques. For example, JP-A-9-15225 describes a gel filtration method for carrying out separation based on molecular sizes, but this method has problems in that it requires a prolonged period of time for separation and a precise separation cannot be made due to the presence of many overlapping parts of lipoprotein peaks. (The term “JP-A” as used herein moans an “unexamined published Japanese patent application”.) On the other hand, a method by an ion exchange chromatography which uses an ion exchanger having a hydrophilic polymer layer covering the surface of porous particles and having functional groups substantially only on said polymer layer is proposed in JP-A-11-180996, but it has disadvantages in that elution of lipoproteins applied to the column is poor and durability of the column is poor. Also, according to the ion exchanger disclosed in JP-A-11-180996, the difference between sodium chloride concentration in the mobile phase at the time of the elution of high density lipoprotein (HDL) and sodium chloride concentration in said mobile phase at the time of the elution of low density lipoprotein (LDL) is small, so that separation of HDL and LDL is not sufficient and it therefore does not reach a level applicable to actual clinical diagnoses in view of analytical accuracy.
On the other hand, it has been revealed that existing amount of modified lipoproteins in blood plasma, particularly increasing degree of oxidized modified LDL (one form of modified LDL) in plasma formed from low density lipoprotein (LDL) by undergoing oxidation reaction in the living body, reflects morbid states more strongly. Accordingly, markedly useful information for clinical findings could be obtained if the increasing degree of oxidized modified LDL in plasma could be examined, but to date there are no analyzing methods which can calculate them accurately and broadly.